INTRODUCTION:

Felodipine (FLD) is used for the treatment of hypertension¹ and acts as calcium antagonist. Felodipine is chemically known as 3 ethyl 5 methyl 4-(2,3-dichlorophenyl)-2,6- dimethyl-1,4-dihydro pyridine -3,5-dicarboxylate. Felodipine is a potent and unique drug that shows fluorescent action. Felodipine is a long-acting 1,4-dihydropyridine calcium channel blocker (CCB)b. It acts primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type calcium channels in their inactive conformation.

By inhibiting the influx of calcium in smooth muscle cells, felodipine prevents calcium-dependent myocyte contraction and vasoconstriction. In the literature survey very few analytical methods were described for the analysis of Felodipine which include spectroscopic techniques², LC-MS/MS³, GC-MS⁴, HPLC methods^5-12 and UFLC^13-15. In the current study a new stability indicating RP-HPLC method for the estimation of Felodipine PR tablets was proposed for the assay of pharmaceutical formulations.

MATERIALS AND METHODS:

Analytes: (Active Pharmaceutical Ingredients):

The API was supplied by Aurobindo Pharma Ltd, Hyderabad.

Chemicals and Reagents:

Trifluoroacetic acid (AR), Water (Milli-Q), Acetonitrile (HPLC grade), Triethylamine (AR), Orthophosphoric acid (AR), 0.22µm (AR), PVDF filter (AR), 0.45µm PVDF filter (AR) were used. Felodipine is existing as PLENDIL® tablets with label claim of 2.5, 5 and 10mg of drug.

Table 1: Chromatographic Conditions

S. No.	PARAMETER	CONDITION
1	Mobile phase	Sodium Dihydrgen Phosphate Monohydrate: Methanol: Acetonitrile (2:2:1)
2	Pump mode	Isocratic
3	Diluent	Sodium Dihydroen Phosphate Monohydrate: Methanol: Acetonitrile (2:2:1)
4	Column	Inertsil ODS _2 C18, 5µ (150*4.6mm)
5	Wavelength	238 nm
6	Column temperature	25° C
7	Injection volume	40µl
8	Flow rate	1.0ml/min
9	Run time	20 min

Validation:^16-19

Linearity:

A Series of solutions are prepared by using stock solution to get concentration levels from 10% to 120% and injected into the chromatographic system. A calibration graph is plotted between concentration of drug (µg/mL) and chromatographic peak area (mV) and Correlation coefficient %RSD was calculated. %RSD of the assay value for six determinations should not be more than 2.0%.

Accuracy:

The standard solutions of accuracy 50%, 100% and 150% were injected into chromatographic system and the individual % recovery and mean % recovery values were calculated

Flow rate:

Standard and Test solutions of 100% concentration was prepared and injected into the HPLC system by keeping flow rates 0.9ml/minand 1.1ml/min

Assay:

40mL of the standard and sample solutions of Felodipine were injected into the HPLC system and the chromatograms were recorded. Amount of drug present in the capsules were calculated using the peak areas.

Degradation conditions:

For acid degradation 5N HCl, 2.1ml is refluxed for 60 minutes at 85˚C temperature. Alkaline and Oxidation studies were performed with 5N NaOH, 30% H₂O₂, in the same environment and these solutions were used injected and chromatograms were recorded.

Effect of heat:

Felodipine peaks should be pure and homogenous. There should not be any interference.

RESULTS:^20-23

Assay:

Method development for the assay of felodipine in tablets was initiated based on general method development guidelines and literature. Several trails were conducted to optimize various parameters and the optimized peak was shown below in fig.no’s 1,2. Mobile phase containing the mixture Sodium Dihydrogen Phosphate Monohydrate: Methanol: Acetonitrile (2:2:1 v/v), with a flow rate of 1.0 ml/min has shown a sharp peak with theoretical plates more than 2000 and tailing factor less than 2.

Fig. No: 1 Chromatogram for the assay of felodipine (standard) in tablets

Fig. No: 2 Chromatogram for the assay of felodipine (sample) in tablets

System suitability:

A Standard solution of Felodipine working standard was prepared as per procedure and was injected five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms obtained by calculating the % RSD of retention time; tailing factor, theoretical plates and peak areas from five replicate injections. The results were tablulated in table no 2. The Tailing factor and %RSD for felodipine are not more than 2.0. The number of theoretical plates (N) for the Felodipine is not less than 1500. All the System suitability parameters were satisfied, thus the method passed the system suitability test.

Fig. No: 3 Chromatogram of system suitability

Table 2: Results of System suitability

S. NO	INJECTION	Area	Plate Count	Tailing Factor
1	Inection_1	1153449	5694	1.1
2	Injection_2	1149534	5627	1.1
3	Injection_3	1150624	5624	1.1
4	Injection_4	1149043	5629	1.1
5	Injecion_5	1148400	5625	1.1
Mean	_	230042
SD	_	1775.245
% RSD	_	0.6

Linearity:

Weigh and transfer accurately about 25mg of Felodipine working standard in a 100ml clean, dry volumetric flask. add 60ml of methanol and sonicate to dissolve. Dilute to volume with methanol and mix. Dilute 4 ml of the solution to 100 ml with diluent and mix .Filter the solution through membrane filter. The calibration curve and the results are as follows.

Table 3: Data of Linearity

Level	Volume of Stock taken (ml)	Dilution (ml)	Concentration (ppm)	Peak Area
10	1.00	250	0.04	121825
25	2.00	200	0.10	305386
50	2.00	100	0.20	604671
75	6.00	200	0.29	909973
100	4.00	100	0.39	1209916
120	4.80	100	0.47	1463432

Fig. No: 4 Calibration Curve for Felodipine

Precision:

It is the quality, condition, or fact of being exact and accurate.

System Precision:

Six working standard solutions of same concentrations were prepared, each injection from each working standard solution was given and the obtained areas were mentioned. Average area, standard deviation and % RSD were calculated. % RSD obtained as 0.11%. as the limit of Precision was less than “2.0” the system precision was passed in this method. The tabular results and the chromatograms are as follows:

Table 4: Data of System precision

S. NO.	Injection	Area
1	Injection-1	1198935
2	Injection-2	1200891
3	Injection-3	1200747
4	Injection-4	1199653
5	Injection-5	1197702
6	Injection-6	1201213
Mean	-	200148.5
Standard Deviation	-	1359.09
% RSD	-	0.11

Fig. No: 5 Chromatogram of System Precision _1

Fig. No: 5.1 Chromatogram of System Precision _2

Fig. No: 5.2 Chromatogram of System Precision _3

Fig. No: 5.3 Chromatogram of System Precision _4

Fig. No: 5.4 Chromatogram of System Precision _5

Fig No: 5.5 Chromatogram of System Precision _6

Method Precision:

It is also called as repeatability/Intra-day precision indicates whether a method gives consistent results for a single batch. Method precision was demonstrated by preparing six test solutions at 100% concentration as per the test procedure and recording the chromatograms of six test solutions. The % RSD of peak areas of six samples was calculated. The method precision was performed. The % RSD of the assay value for six determinations should not be more than 2.0%. The tabular results and the chromatograms are as follows:

Table 5: Data of Method precision

S. No.	Injection	Area of felodipine
1.	Injection_1	909494
2.	Injection_2	914264
3.	Injection_3	906609
4.	Injection_4	907809
5.	Injection_5	909702
6.	Injection_6	911371
Mean	_	151645.8
S. D	_	2705.976
%RSD	_	0.3

Fig No: 6 Chromatogram of Method Precision _1

Fig No: 6.1 Chromatogram of Method Precision _2

Fig No: 6.2 Chromatogram of Method Precision _3

Fig No: 6.3 Chromatogram of Method Precision _4

Fig No: 6.4 Chromatogram of Method Precision _5

Fig No: 6.5 Chromatogram of Method Precision _6

Accuracy:

The standard solutions of accuracy 50%, 100% and 150% were injected into chromatographic system. The % recovery for each level is between 98.0 to 102.0%.

Robustness:

Robustness conditions like low flow (0.9ml/min), high Flow (1.1ml/min), low mobile phase, high mobile phase, low temperature (23°C) and high temperature (27°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit. The LOD Value for Felodipine was found to be 0.56 µg/ml respectively. The LOQ Value for Felodipine was found to be 1.71µg/ml respectively.

Table 7: Results of LOD, LOQ

S. No.	Sample	LOD	LOQ
1.	Felodipne	0.56µg/ml	1.71 µg/ml

Degradation conditions:

Felodipine peaks should be pure and homogenous. There should not be any interference.

The degradation studies are tabulated as follows:

· Acid Degradation: 5N HCl, 2.1ml for 60 minutes at 85˚C temperature.

· Base Degradation: 5N NaOH, 2.1ml for 60 minutes at 85 ˚C temperature.

· Peroxide Degradation: 30% H₂O₂, 2.1ml for 60 minutes at room temperature.

· Thermal Degradation: Heated at 70 ˚C for 12 hrs.

Fig No: 7 Chromatogram of acid degradation

Fig No: 7.1 Chromatogram of base degradation

Fig No: 7.2 Chromatogram of peroxide degradation

Fig No: 7.3 Chromatogram of thermal degradation

Table 8: Data of Degradation

S. No.	Type of degradation	% Assay	% Degraded
1.	Acid	95.30	4.70
2.	Base	97.27	2.73
3.	Peroxide	98.01	1.99
4.	Thermal	99.27	0.73

CONCLUSION:

The study was focused to develop and validate RP-HPLC method for simultaneous estimation of felodipine in its oral formulation. The developed method was useful for routine analysis and is capable of analyzing huge number of samples in a short period with good robustness, accuracy and precision. This RP-HPLC method generates large amount of quality data, which serve as highly powerful and convenient analytical tool. From the experimental data obtained it was concluded that, the chromatographic method developed for estimation of felodipine was found to be simple, precise, accurate, sensitive and cost effective with good reproducibility and recovery. All the parameters were validated as per ICH guidelines and found to be within the acceptance criteria. So the developed method may be recommended for routine analysis in research institutions and quality control departments in industries for the determination of felodipine its oral formulations.

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Received on 20.05.2020 Modified on 13.06.2020

Asian J. Pharm. Ana. 2020; 10(4):207-212.

DOI: 10.5958/2231-5675.2020.00038.1

% Concentration	Area	Amount Added (mg)	Amount Found	% Recovery	Mean Recovery (%)
50%	610589	10.29	10.06	98.13	98.7
100%	1221087	20.27	20.10	99.56
150%	1785799	30.00	29.43	98.52