A VALIDATED, STABILITY-INDICATING HPLC METHOD FOR THE DETERMINATION OF FELODIPINE AND ITS RELATED SUBSTANCES

A VALIDATED, STABILITY-INDICATING HPLC METHOD FOR THE DETERMINATION OF FELODIPINE AND ITS RELATED SUBSTANCES

Liandong Hu ^{*1, 2}, Qiaofeng Hu ^{1, 2} and Na Gao ^{1, 2}

School of Pharmaceutical Sciences, Hebei University ¹, PR China

Key Laboratory of Pharmaceutical Quality Control of Hebei Province ², Baoding, PR China

ABSTRACT: An HPLC method was developed and validated to determine felodipine and related substances. A C18 column (5μm, 250 ×4.6 mm) was used for the separation at room temperature, with methanol: acetonitrile: water (50:15:35%, v/v/v) as the mobile phase at the flow rate of 1.0mLmin⁻¹. The detection wavelength was 238 nm. The validation characteristics included accuracy, precision, specificity, linearity, and robust, and stability-indicating. The method showed good linearity for felodipine and its related substances with correlation coefficients in the range of 5.05-40.4μg/mland 0.31-15.50μg/ml. Method accuracy was assessed for felodipine and its related substances at three levels, the recovery ranged from 98.86 to 101.03%. The intermediate precision were 0.42% for felodipine and was 1.01% for the related substances (n = 9). Limit of detection and quantification for felodipine and its related substances were 1 and 4ng, respectively. The solution remains stable within eight hours at room temperature. Finally, the method was demonstrated to be robust, resistant to small variations of chromatographic variables. This method is fast, simple, can be used for direct determination of felodipine and its related substances in the pharmaceutical preparation.

INTRODUCTION:  Hypertension  is  a  major  risk  factor  for arteriosclerosis and the beneficial effects of lowering blood  pressure  on  the  vascular  morbidity  and mortality are well documented and demonstrated. Felodipine, a new generation calcium channel antagonist, belonging to the class of dihydropyridines, is a practical advance in the treatment of hypertension ¹.

Felodipine is a highly vasoselective calcium antagonist that effectively reduces arterial blood pressure ².

It does not have any negative inotropic effects at doses used for antihypertensive therapy ^{3, 4}. It has a direct relaxing effect on smooth muscle cells, especially those of resistance vessels.

Clinical studies have shown that felodipine is an effective drug for the treatment of essential hypertension ^5-7.

There are impurities/related substances associated with the manufacture of felodipine drug substance. The process related impurities are observed with synthetic routes and/or manufacturing processes.

Structures of felodipine and its related substances and their chemical names are provided in Table 1. These related substances are monitored during the release of drug substance raw material and finished drug products.

The primary goal of this study was to develop and validate an HPLC method that could separate felodipine from its potential related substances and has sufficient sensitivity for quantitation of these impurities at very low concentrations. The present analytical method discussed in this article is a simple isocratic high performance liquid chromatography with ultraviolet detection for the determination of felodipine and its related substances.

TABLE 1: CHEMICAL NAMES AND STRUCTURES FOR FELODIPINE AND ITS RELATED SUBSTANCES IMPURITY I

Materials and reagents: Methanol, acetonitrile (HPLC grade) and phosphate buffer was obtained from Tianjin Kermel Chemical Reagent Co. Ltd (Tianjin, China).Standard Felodipine (99.9% of purity) and Impurity I (ethyl methyl4-(2,3-dichlorophenyl)-2,6- dimethylpyridine-3,5-dicarbo xylate) (98.6% of purity)were obtained fromThe National Institute for Control of Pharmaceutical and Biological Products. All other chemicals were of analytical grade. Water was purified by redistillation and passed through a 0.22μm membrane filter before use. EXPERIMENTAL:

HPLC conditions ⁸: The HPLC analysis system consisted of a LC-20AT liquid chromatogram and SPD-20A UV/VIS detector (Shimadzu, Kyoto, Japan) and the chromatographic column was a Kromasil C-18 (5μm, 250 ×4.6 mm).Mixture of methanol: acetonitrile: water (50:15:35%, v/v/v) was used as the mobile phase. The flow rate was 1.0 ml/min; UV-detection was at a wavelength of 238 nm.

Sample preparation:10 tablets were weighed and finely powdered. A portion of powder equivalent to felodipine 5 mg was accurately weighed into 50 ml volumetric flasks and 40ml ethanol was added. The volumetric flasks were sonicated for 5min, then the solutions were then made up to volume with ethanol, shaken, centrifugal, take supernatant fluid, the solution was filtered through 0.45μm filter.

Accurately measuring continued filtrate 5m1, put it into 25 ml volumetric flasks, the solutions were then made up to volume with mobile phase, shaken, 20μl of the test solution was injected and chromatogram was recorded; then accurately weighed felodipine reference substance drying to constant at 105^oC, the solutions were then dissolved and made up to per 1ml contains 20μg samples with mobile phase, and determined with the same method, calculated peak area with external standard.

Related substances preparation: Impurity I reference substance were weighed, methanol was added to dissolve and quantitative dilution made 1ml solution contains 0.3mg, then 1ml was accurately measured and transferred to a 100ml volumetric flask, 1 ml test solution was added and then diluted to volume with the mobile phase, and mixed.

RESULTS AND DISCUSSION:

 Method validation: The method was validated according to the high performance liquid chromatography (the Chinese pharmacopoeia 2010 edition appendix V D).

The following validation parameters were addressed: linearity, range, accuracy, precision, specificity and limit of detection, limit of quantitation, solution stability destructive and robustness.

 System suitability: System suitability tests were performed to ensure that the HPLC system and procedure are capable of providing quality data.All those parameters reported in Table 2.

TABLE 2: VALIDATION OF THE METHOD REGARDING SYSTEM SUITABILITY, LINEARITY, LIMITS OF DETECTION AND QUANTITATION.

Linearity and range: The linearity of felodipine and Impurity I were evaluated at five concentrations in the range of 5.05-40.4μg/ml and 0.31-15.50μg/ml,respectively. The linearity curves were defined by the following equations

y =64193 x - 2218，R²= 0.999 for felodipine

y = 34701x+3825.2，R² = 0.9994 for Impurity I

Where y is the peak area of analyte and x is the analyte concentration. The results show excellent correlations between peak area and concentration over the desired concentration range.

Accuracy and Precision: Accuracy and precision were established across the analytical range for felodipine. Accuracy of the method was calculated by recovery studies by standard addition method. The method was determined by preparing the samples of the same batch in nine determinations with three concentrations and three replicate each. The accuracy of the method is shown in Table 3.The precision is shown in Table 4.

TABLE 3:  RESULTS OF ACCURACY

   TABLE 4:  INTERMEDIATE PRECISION OF FELODIPINE AND IMPURITY I

4. Specificity: The specificity of the HPLC method is illustrated in Fig. 1, Impurities I were spiked and were found to be well separated from felodipine.
5. 

Stability: Sample solution was performed after they were stored at room temperature and under refrigerated conditions over time. All solutions are stable for at least 8h at room temperature, the data was shown in Table 5, and the result indicated that the solution was stable in 8 hours at room temperature.

5. Destruction test: In order to determine the specificity of felodipine, a raw materials destruction test was carried out. The test involved exposure to acid, base, light, H₂O₂ (oxidative medium) and heat. Fig. 2 shows the chromatograms of felodipine raw materials after treatment with acid, base, light, heating and oxidation by H₂O₂. It can be seen that the related substances can be detected and separated effectively.
6. Robustness: The robustness of the method was examined by small variations of critical parameters, such as changes of pH of the mobile phase, flow rate, percentage of acetonitrile in the mobile phase. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 6).

TABLE 5   STABILITY OF FELODIPINE SAMPLES AND IMPURITY I UNDER INDICATED CONDITIONS

FIG. 2: REPRESENTATIVE CHROMATOGRAMS OF FELODIPINE RAW MATERIAL AFTER TREATMENT WITH 0.1MOL/L HYDROCHLORIDE (A); 0.1MOL/L SODIUM HYDROXIDE (B); LIGHT(C);HEAT (D); H₂O₂ (E).

TABLE 6:  RESULTS OF ROBUSTNESS STUDY

CONCLUSIONS: An HPLC method has been developed and validated for the determination of felodipine and related substances. The developed method is accurate, precise and linear across the analytical range. The method is stability-indicating and specific for the determination and quantitation of trace levels of felodipine and its related substances. This method has especially low limits of detection and quantitation, and was proven to be robust, accurate and precise.

The method is therefore suitable for felodipine and its related substances and applied to other pharmaceutical dosage forms.

ACKNOWLEDGEMENT: This work was supported by the Talent Introduction Program of Hebei University (No. y2005064), the Medical and Engineering Science Research Center of Hebei University (No. BM201109). Hebei Provincial Natural Science Foundation of China- Shijiazhuang Pharmaceutical Group (CSPC) Foundation (No. H2013201274).

REFERENCES:

 Walash MI and Belal FF: Synchronous fluorescence spectrofluorimetric method for the simultaneous determination of metoprolol and felodipine in combined pharmaceutical preparation. Chem Cent J 2011; 5: 70.

1. Bazzo GC and Caetano DB: Enhancement of felodipine dissolution rate through its incorporation into Eudragit® E-PHB polymeric microparticles: in vitro characterization and investigation of absorption in rats. J Pharm Sci 2012; 101: 1518-1523.
2. Hsiao CL and Wu YC: Pharmacokinetics of felodipine extended-release tablets in healthy Taiwanese subjects: a retrospective review. Arzneimittelforschung 2011; 61: 444-451.
3. Palem CR and Kumar Battu S: Role of cyclodextrin complexation in felodipine-sustained release matrix tablets intended for oral transmucosal delivery: in vitro and ex vivo characterization. Pharm Dev Technol 2012; 17: 321-332.
4. Al-Niaimi F and Lyon C: Felodipine-induced eruptive telangiectasia following mastectomy and radiotherapy. Br J Dermatol 2010; 162: 210-211.
5. Edgar B, Lundborg P, Regardh CG. Clinical Pharmacokinetics of felodipine, a summary. Drugs. 1987; 34: 16-27.
6. Dunselman PH, Edgar B. Felodipine clinical pharmacokinetics. Clinical Pharmacokinetics 1991; 21: 418-430.
7. Chinese Pharmacopoeia Commission Office: Felodipine tablets. Chinese Pharmacopoeia, 2010; 454-455.