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.Development and validation of UV spectrophotometric method for estimation of process related impurity in felodipine bulk and formulation

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  • Rashtriya Shikshan Mandals N.N. Sattha College of Pharmacy Ahmednagar

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This research is directed towards synthesis and characterization of process related impurity of Felodipine i.e.diethyl 4-(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (FI) in bulk and tablet formulation by UV, IR and NMR techniques and its quantitationby UV spectrophotometric method development. The synthesis of (FI) was carried out by Hantzch process using p-chlorobenzaldehyde, ethylacetoacetate in presence of ammonia and methanol as catalyst. The preliminary evaluation was done on laboratory scale viz. melting point, TLC and elemental analysis. The regression coefficient was found to be 0.999 and Relative Standard Deviations were below 2%. The method was validated as per ICH guidelines and was found to be linear, precise, accurate, robust and rugged.
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Scholars Research Library
Der Pharmacia Lettre, 2015, 7 (4):284-290
(http://scholarsresearchlibrary.com/archive.html)
ISSN 0975-5071
USA CODEN: DPLEB4
284
Scholar Research Library
Development and validation of UV spectrophotometric method for estimation
of process related impurity in felodipine bulk and formulation
Vaibhav M. Thorat*
1
, Pawar S. S.
2
, Pande V. V.
3
, Arote S. R.
4
and Musmade Deepak S.
2
1
Department of Quality Assurance Techniques, S.R.E.S, Sanjivani College of Pharmaceutical Education and
Research, Kopargaon, Maharashtra, India
2
Department of Pharmaceutical chemistry, S.R.E.S, Sanjivani College of Pharmaceutical Education and Research,
Kopargaon, Maharashtra, India
3
Department of Pharmaceutics, S.R.E.S, Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
4
Department of Pharmacology, S.R.E.S, Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
_____________________________________________________________________________________________
ABSTRACT
This research is directed towards synthesis and characterization of process related impurity of Felodipine i.e.diethyl
4-(4-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (FI) in bulk and tablet formulation by UV,
IR and NMR techniques and its quantitationby UV spectrophotometric method development. The synthesis of (FI)
was carried out by Hantzch process using p-chlorobenzaldehyde, ethylacetoacetate in presence of ammonia and
methanol as catalyst. The preliminary evaluation was done on laboratory scale viz. melting point, TLC and
elemental analysis. The regression coefficient was found to be 0.999 and Relative Standard Deviations were below
2%. The method was validated as per ICH guidelines and was found to be linear, precise, accurate, robust and
rugged.
Keywords: Felodipine, Hantzch process, Impurity, Spectrophotometric analysis
_____________________________________________________________________________________________
INTRODUCTION
Felodipine chemically is Ethyl methyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydro-3,5-
pyridinedicarboxylatewith molecular formula C
18
H
19
Cl
2
NO
4
and molecular weight 284.3 [1]. Felodipine is under
class of Calcium Channel Blocker used in the treatment of myocardial infraction, heart failure [2, 3].Felodipine
decreases arterial smooth muscle contractility and subsequent vasoconstriction by inhibiting the influx of calcium
ions through voltage-gated L-type calcium channels [4].
UV Spectrophotometric method was developed using methanol as solvent. The developed method was optimized
and validated as per guidelines of International Conference on Harmonization (ICH) According to ICH guidelines
on impurities in new drug product, when the impurity is less than 0.1% level it is not considered to be necessary,
unless impurities found to be toxic or potent [5,6].
Vaibhav M. Thorat et al Der Pharmacia Lettre, 2015, 7 (4):284-290
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Figure 1: Chemical structure of FI
MATERIALS AND METHODS
Chemicals
P-chlorobenzaldehyde (AR), Ethylacetoacetate (AR), Ammonia (AR), Methanol (AR) were purchased from Merck
Chemicals, India.
Synthesis of Felodipine Impurity
The synthesis of Felodipine Impurity (FI) was carried out by addition of 0.01 mole of p- chlorobenzaldehyde,
0.02moles ethylacetoacetate, 3 ml ammonia, 15 ml methanol and was refluxed for 8 hours. Then it was cooled,
poured into 150 ml ice cold waterand stirred for 1 hour. Then it was filtered, dried and recrystallized twice using
methanol as solvent and weighed.
Figure 2: Synthesis of FI
FT-IR
The IR spectrum was recorded using KBr press pellet technique by using Fourier Transform Infrared
Spectrophotometer Model No. 8400S SHIMADZU INC.
Vaibhav M. Thorat et al Der Pharmacia Lettre, 2015, 7 (4):284-290
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NMR
The characterization of impurity was done by using NMR. The
1
H and
13
C NMR were reported by using CDCl
3
as
solvent.
UV
Determination of wavelength of maximum absorption
Accurately weighed 10 mg of FI was transferred to 100 ml volumetric flask and volume was made upto 100 ml with
methanol. The solution was scanned from 200 – 400 nm to determine λmax.
Linearity and Range
The aliquots of stock solution of FI (0.2, 0.4, 0.6, 0.8, 1.0) were transferred to 10 ml volumetric flask and volume
was made up to 10 ml by methanol for making 2ppm, 4ppm, 6ppm, 8ppm and 10 ppm. The absorbance of solution
was taken at 237 nm against methanol as a blank.
Precision
In intra-day precision, two repeated readings after four hours were taken and % RSD was calculated. In inter-day
precision two repeated measurement were made on two consecutive days and % RSD was calculated.
LOD and LOQ
Detection limit and Quantification limit was calculated using formula
LOD= 3.3 × SD/ Slope
LOQ=10 × SD/Slope
Where, SD is calculated using values of y intercepts of regression equations.
Robustness
Robustness was studied by changingscanning speed. The SD and % RSD between the changed parameter was
calculated.
Ruggedness
Ruggedness was studied by changing analyst. The SD and % RSD between the changedanalysts was calculated.
Accuracy and Recovery
To ensure the accuracy, known amounts of pure drug (50%, 100%, and 150%) were added to the sample solution
and these samples were reanalysed by the proposed method and also % recovery was determined.
RESULTS AND DISCUSSION
Physicochemical properties
Table no 1: Physicochemical properties
Molecular formula Molecular weight M.P
o
C Rf value
Benzene: Methanol
(6:1 v/v) % yield
C
19
H
22
ClNO
4
363.5 136-140 0.64 75%
Thin layer chromatography (TLC)
Rf value = 0.64
IR data[6,7,8]
The major functional groups are primary amine, chloro and carbonyl groups. Obtained peaks in IR spectrum are as
follows.
IR (KBr) : 3354.32 (NH- Stretch), 2958.90,3088.14 (C-H Aromatic Stretch), 2899.11 (C-H Aliphatic
Stretch), 1695.49 (C=O), 1489.10 (C=C), 1375.29 ( Bend), 1174.69-1215.19 (C-O-C Stretch), 746.48-783.13
(Benzene ring Bend), 831.35 (CH out of plane bending of para-benzoid), (Substitution at para position of benzene
ring)
Vaibhav M. Thorat et al Der Pharmacia Lettre, 2015, 7 (4):284-290
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NMR data [6,8]
1
H NMR (CDCl
3)
δ (ppm)= 4.945 (1H,NH of 1,4 dihydropyridine), 1.206 (6H,CH
3
of 1,4 dihydropyridine), 4.021 (4H, CH
2
proton of
ester), 2.28 (6H,CH
3
proton of ester), 6.120 (1H,CH of 1,4 dihydropyridine), 7.141 (2H,CH of chlorobenzene ring),
7.207 (2H,CH of chlorobenzene ring).
13
C NMR (CDCl
3
)
δ (ppm)=14.16 (2C,CH
3
Carbon attached to CH
2),
50.94 (2C,CH
2
Carbon attached to CH
3
), 167.93 (2C, Carbonyl
carbon attached to 1,4-dihydropyridine ring), 19.34 (2C,CH
3
Carbon attached to 1,4-dihydropyridine ring), 127.91
(2C,C=C of 1,4-dihydropyridine ring), 129.29 (2C,C=C of 1,4-dihydropyridine ring), 38.94 (1C, Carbon of 1,4-
dihydropyridine ring), 144.30 (6 Carbon of phenyl ring).
UV method development
UV Spectrum [7, 9, 10]
A B
C
Figure 3: UV spectrum of A- Felodipine, B- FI, C- Overlay of Felodipine and FI
Felodipine
FI
Vaibhav M. Thorat et al Der Pharmacia Lettre, 2015, 7 (4):284-290
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Table no 2: Linearity
Sr. no Concentration
(ppm) Absorbance
1 2 0.1191
2 4 0.1725
3 6 0.2726
4 8 0.3848
5 10 0.5078
6 12 0.5931
7 14 0.6787
8 16 0.7850
9 18 0.8973
Figure 4: Calibration curve
Table no 3: Intra-day Precision
Sr. no Concentration
(ppm) Absorbance SD %RSD
1 6 0.2969
0.0065 2.0
2 6 0.2992
3 6 0.3077
4 6 0.2922
5 6 0.3070
6 6 0.2913
7 6 0.3032
Table no 4: Inter-day Precision
Sr. no Concentration
(ppm) Absorbance SD %RSD
1 6 0.3462
0.0026 0.77
2 6 0.3429
3 6 0.3512
4 6 0.3530
5 6 0.3478
6 6 0.346
6 6 0.3454
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Table no 5: Ruggedness
Sr. no Concentration
(ppm) Absorbance
1 Absorbance
11 SD
1 SD
11 %RSD
1 %RSD
11
1 6 0.2986 0.2986
0.005425 0.005808 1.84 1.99
2 6 0.2972 0.2806
3 6 0.2854 0.2913
4 6 0.2961 0.2886
5 6 0.2869 0.2901
6 6 0.2916 0.2954
7 6 0.2979 0.2955
Table no 6: Robustness
Sr. no Concentration
(ppm) Absorbance
1 Absorbance
11 SD
1 SD
11 %RSD
1 %RSD
11
1 6 0.3050 0.3099
0.003714 0.0033 1.22 1.05
2 6 0.3066 0.3105
3 6 0.2979 0.3123
4 6 0.2991 0.3085
5 6 0.3056 0.3175
6 6 0.3059 0.3154
7 6 0.3070 0.3151
Table no 7: Recovery
Sr. no Drug / Formulation Percentage recovery Mean SD %RSD
50% 100% 150%
1 Bulk 95.69 98.78 99.32 97.93 1.95 1.99
2 Tablet 96.0 97.4 99.29 97.56 1.63 1.67
Method Validation
Linearity and Range
The given method was obtained in range of 2-18 µg/ml. The standard Calibration curve was obtained by plotting the
absorbance against its concentration measured at 237 nm. The regression coefficient was found to be 0.999 and
slope was found to be 0.0498
Intra-day and Inter-day Precision
The intra-day and inter-day precision study of the developed method confirmed adequate sample stability and
method reliability where all the Relative Standard Deviations were below 2%.
Ruggedness
The method was performed by changing analyst and the method was found to be rugged with standard deviation
0.005808 and relative standard deviation 1.99%.
Robustness
The robustness was performed by change in scanning speedand method was robust with standard deviation 0.0033
and relative standard deviation 1.05%.
LOD and LOQ
The LOD 0.2650and LOQ 0.8835ensures that the method is more sensitive and selective.
Accuracy and Recovery
The results within the range 96.00-99.00 ensure an accurate method.
CONCLUSION
The synthesis of a process-related impurity of Felodipine was successfully carried out by suitable synthetic
procedure. Its characterization was carried out by IR,
1
H NMR and
13
C NMR. The result and statistical data states
that the UV spectrophotometric method was found to be linear, precise, robust, rugged and accurate as per ICH
guidelines.
Acknowledgement
Author wish to express their sincere thanks to Principal, SRES’s Sanjivani College of Pharmaceutical education and
research, Kopargoan for his constant encouragement and support.
Vaibhav M. Thorat et al Der Pharmacia Lettre, 2015, 7 (4):284-290
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REFERENCES
[1] British Pharmacopoeia, The Department of Health, British Pharmacopoeia Commission Office,volume I,2011,
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2008,6, 181-182.
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2014, 4, 2231-6876, 181-188.
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... At present, few studies have been reported on the use of RP-HPLC [18], UV-VIS [19], and HPTLC [20] for the determination of FEL in bulk drugs or pharmaceutical preparations. Although UV-visible spectrophotometry has the advantages of simple operation, the accuracy cannot satisfy the requirements of experiments, and the sample concentration has high requirements, which are relatively difficult to attain, and the volumetric analysis is poor in sensitivity. ...
Article
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In this work, a CdTe quantum dot-based fluorescent probe was synthesized to determine felodipine (FEL). The synthesis conditions, structure, and interaction conditions with FEL of CdTe quantum dots were analysed by fluorescence spectrophotometry, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), UV–visible spectroscopy, and TEM. The CdTe QD concentration was 2.0 × 10−4 mol/L. The amount of quantum dots controlled in the experiment was 0.8 mL. The controlled feeding ratio of N (Cd2+):N (Te2−):N (TGA) was 2:1:4, the heating temperature was 140 °C, the heating time was 60 min, and the pH of the QD precursor was adjusted to 11 for subsequent experiments. The UV–visible spectrum showed that the emission wavelength of CdTe quantum dots at 545 nm was the strongest and symmetric. The particle size of the synthesized quantum dots was approximately 5 nm. In the interaction of CdTe quantum dots with FEL, the FEL dosage was 1.0 mL, the optimal pH value of Tris-HCl buffer was 8.2, the amount of buffer was 1.5 mL, and the reaction time was 20 min. The standard curve of FEL was determined under the optimal synthesis conditions of CdTe quantum dots and reaction of CdTe quantum dots with FEL. The linear equation was Y = 3.9448x + 50.068, the correlation coefficient R2 was 0.9986, and the linear range was 5 × 10−6–1.1 × 10−4 mol/L. A CdTe quantum dot-based fluorescent probe was successfully constructed and could be used to determine the FEL tablet content.
... In literature, very few RP-HPLC [4][5][6][7][8], UV-Spectrophotometric [9] and HPTLC [10], methods have been reported for determination of felodipine in bulk drugs and pharmaceutical formulations and also in combination with other drugs. Hence an attempt has been made to develop a new assay method with less retention time for protein binding study of felodipine. ...
Article
Objective: The aim of present study was to develop and validate a new simple, easy, selective, precise, accurate reverse phase high-performance liquid chromatography for the estimation of felodipine in bulk and pharmaceutical dosage form.Methods: The separation was carried on HPLC system consisting C18 column (150 mm ×4.6 nm, 5 µm) at room temperature coupled with a phenomenixcolumn silica with flow rate 1 ml/min. The mobile phase used was methanol: acetonitrile in the ratio of 50: 50. The drug was detected using UV-visible detector at the wavelength of 230 nm and run time was 10 min.Results: The retention time was 3.138 min. Linearity was observed in the concentration range of 5-25μg/ml. The accuracy of the method was assessed by percentage recovery studies at three different levels at 80%, 100% and 120% of its working concentration. The percentage recovery of felodipine in the developed method was found to be in the ranges of from 99.81-100.00% that indicates the good accuracy of the method. The percentage % RSD of precision was found to be less than 2%. The method was validated as per ICH guidelines. The developed method was employed in in vitro protein binding studies using semi permeable membrane and performed by plotting calibration curve (peak area vs concentration) the % drug release of felodipine was calculated.Conclusion: The proposed method was found to be simple, precise, accurate and consistent. The validated parameters are statistically validated for linearity, precision and limit of detection, limit of quantification, robustness, ruggedness were concluded.Â
The Department of Health, British Pharmacopoeia Commission Office,volume I
  • British Pharmacopoeia
British Pharmacopoeia, The Department of Health, British Pharmacopoeia Commission Office,volume I,2011, 1, 776-778.
  • K D Tripathi
K.D. Tripathi, Essential of Medical Pharmacology, Jaypee Brothers Medical Publishers (P) Ltd, New Delhi; 2008,6, 181-182.
  • V Shinde
  • S Gosavi
  • D Musmade
  • S Pawar
  • V Kasture
V. Shinde, S. Gosavi, D. Musmade, S. Pawar, V. Kasture,Indo American Journal of Pharmaceutical Research, 2014, 4, 2231-6876, 181-188.
  • G P Jadhav
  • V S Kasture
  • S S Pawar
  • A P Lodha
  • A R Vadgaonkar
  • R K Ajage
  • S G Deshpande
G.P. Jadhav, V.S. Kasture, S.S. Pawar, A.P. Lodha, A.R. Vadgaonkar, R.K. Ajage, S.G. Deshpande, International Journal of Pharmacy and Pharmaceutical Sciences, 2014, 6, 7, 401-402
  • D A Skoog
  • D M West
  • F J Holler
  • S R Crouch
D.A.Skoog., D.M.West, F.J.Holler, S.R.Crouch, Fundamentals of Analytical Chemistry, Thomson Brooks/cole, Singapore, 2004,8,906-946.
Spectrometric Identification of Organic Compounds
  • R M Silverstein
  • F X Webster
R. M.Silverstein, F. X.Webster, Spectrometric Identification of Organic Compounds, John Wiley and Sons publications, 2005,6, 81-109.
Handbook of Instrumental Techniques for Analytical Chemistry,Pearson Education
  • F Settle
F. Settle, Handbook of Instrumental Techniques for Analytical Chemistry,Pearson Education, 1997, 481-499.