Stability Indicating RP-HPLC Method for the Determination of Fenoverine in Bulk and Formulation

 

A. Suganthi* and T.K. Ravi

College of Pharmacy, SRIPMS, 395, Sarojini Naidu Road, Coimbatore-641044, Tamilnadu, India

*Corresponding Author E-mail: suganlemu@gmail.com

 

ABSTRACT:

A simple, sensitive and precise RP-HPLC method was developed and validated for the determination of fenoverine (antispasmodic) in presence of its degradation products. Fenoverine and all the degradation products were resolved on a C18 column with the mobile phase composed of methanol, acetonitrile and 10mM ammonium formate (70:10:20, v/v/v) at 258 nm using a photodiode array detector. The method was linear over the concentration range of 550 g mL1 and precise with RSD < 2 % in intra- and inter-day study. Excellent recoveries of 100.571.692 to 101.400.6145 proved the accuracy of the method. Developed method was specific, as indicated by chromatographic resolution > 2.0 for each peak and sensitive with LOD 50 ng mL-1 and LOQ 0.5 g mL1. The method was used to study the drug degradation behavior under forced conditions. Two degradation products were formed during the degradation study in 6% H2O2 and water whereas only one degradation product in 1 mol L1 HCl, 0.01 mol L1 NaOH and photolytic degradation. The method was applied successfully for the assay of fenoverine in the capsule dosage form.

 

KEYWORDS: Fenoverine, high performance liquid chromatography, stability, forced degradation.

 


INTRODUCTION:

The International Conference on Harmonization (ICH) guidelines Q1A (R2) recommend the use of a validated stability-indicating assay method (SIAM) for stability testing of a drug substance or product1. It also emphasizes the conduct of a forced degradation study on the drug substance to generate information on degradation products that can form under the influence of hydrolytic, oxidative, thermal or photolytic degradation conditions. Fenoverine (FEN) (2-[4-(benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl]-1-(10H-phenothiazin-10-yl)ethanone) (Fig 1) is an antispasmodic drug which is known to inhibit contraction of smooth muscles elicited either by electrical stimulation or by potassium depolarization2. Methods of analysis of fenoverine in human serum, tissue samples, plasma and capsule dosage form by HPLC were reported previously3-5. No stability indicating assay of FEN in bulk and solid dosage form could be traced in the literature. In the present study, we aimed to develop and validate a stability indicating RPHPLC assay method that allowed resolution, detection and estimation of fenoverine in the presence of degradation products obtained during the forced conditions in bulk substance and capsule dosage form.

 

Fig1:Chemical structure of fenoverine (FEN)

 

MATERIALS AND METHODS:

Chemicals and reagents:

Fenoverine was kindly provided as a gift sample by Microlabs Pharmaceuticals Ltd, Bangalore, India with not less than 98% purity. Capsule dosage form of fenoverine (Spasmopriv, MicrolabsLtd, India, 100 mg) was purchased from a local pharmacy. HPLC grade methanol and acetonitrile were purchased from S.D. Fine Chemical, India. Hydrochloric acid, sodium hydroxide pellets, hydrogen peroxide solution, and ammonium formate were also purchased from S.D. Fine Chemical and were of analytical grade. Water for RP-HPLC was purchased from Merck, India.

 

Instruments:

The Shimadzu (Japan) Liquid Chromatography Mass Spectrometer system (LCMS-2010EV) was equipped with a diode array detector (SPD-M20A), auto sampler (SIL-20AC) and column oven CTO-10AS vp. Chromatographic separations were performed using the LichroCART 250-4 RP-18 e (5 m particle size) HPLC- cartridge at ambient temperature and analyzed by LCMS solution software Shimadzu. Hot air oven (Sunstar Scientific, India) capable of controlling the temperature within 2C was used for the thermal degradation studies.

 

Preparation of standard solutions:

A stock solution of fenoverine was prepared by dissolving the drug in methanol to get a concentration of 1000g mL1. Fresh stock solution was prepared every day during the experiment. From this stock solution, ten calibration standards (5-50 g mL1) and three quality control samples (5, 20 and 40 g mL1) were prepared by diluting appropriate aliquots of standard solutions.

 

Preparation of sample solution:

Twenty capsules were weighed and emptied. Powder equivalent to 10 mg drug was then transferred to a 100 mL volumetric flask containing methanol. The mixture was then sonicated for 20 min to dissolve the material completely and centrifuged at 3,000 rpm for 5 min. An aliquot of supernatant solution was diluted appropriately to give a solution of 1000 g mL1and 10 mL of this solution was used for forced degradation studies.

 

Forced degradation studies:

Forced degradation of drug substance and drug product was carried out under acid/neutral/basic hydrolytic, oxidative, thermolytic, and photolytic stress conditions. For hydrolytic and oxidative degradation, drug solutions were prepared with a concentration of 500 g mL1. After degradation, aliquots were diluted with methanol to achieve a concentration of 50 g mL1. Methanol was used for drug solubilization in acidic, neutral and oxidative media. Hydrolytic degradation studies were carried out under acid (1 mol L1 HCl, pH 1.07), basic (0.01 mol L1 NaOH, pH 12.1) and neutral (water, pH 6.7) conditions at 70C as well as at room temperature over 12 h. Oxidative degradation was carried out in a 6% H2O2 solution at room temperature over 24 h. Thermal and photo degradation of drug substance and drug product were carried out in solid state. For thermal degradation, the drug was spread in a borosilicate glass Petri dish and placed in the hot-air oven maintained at 80C for 5 h. Also, photolytic studies were carried out by exposing a thin layer of the solid drug in a Petri dish under direct sunlight for 5h. After degradation, stock solutions were prepared by dissolving the samples in methanol to achieve a concentration of 500 g mL1. From these solutions, aliquots were diluted with methanol to get the final concentration of 50 g mL1 of FEN. Samples were withdrawn initially, subsequently at prefixed time intervals and stored in refrigerator until analysis for all forced conditions.

 

Method development:

Detection wavelength for HPLC study was selected as 258 nm after recording the UV spectrum of the drug from 190 to 400 nm. The maximum area and peak selectivity of FEN was observed at this wavelength. The chromatographic conditions were optimized for resolution of the peak of the drug and degradation products under each forced condition by varying the proportion of methanol/acetonitrile/10 mM ammonium formate in the mobile phase and the flow rate using representative samples from each forced condition. Several trials using various proportions of methanol and 10 mM ammonium formate as mobile phase were carried out. However, to attain good peak shape, minimal tailing and selective resolution of FEN and its degradation products, acetonitrile was introduced as the third solvent. An appropriate blank was injected before the analysis of all forced samples. Such an optimized method was then used to study the forced degradation behavior of FEN and was also applied in the stability indicating assay of FEN capsules.

 

Validation of the method:

The optimized method was validated in accordance with the ICH guidelines6. Linearity was determined by analyzing, in triplicate, standard drug solutions of concentrations 5-50 gmL1 using 20 L of the injection volume. For intra-day precision, three quality control drug concentrations (5, 20 and 40 g mL1) were analyzed six times on the same day whereas the same drug concentrations were analyzed on three different days for inter-day precision. Accuracy/recovery was evaluated by spiking the mixture of degradation samples with three known drug concentrations and calculating the percent recovery from the differences between the peak areas obtained for concentrated and diluted solutions. Signal-to-noise ratios were employed to estimate limits of detection (3:1) and limits of quantitation (10:1). The specificity of a method is its suitability for analysis of a substance in presence of potential impurities. Specificity of the method was established through the study of the resolution (Rs) of FEN samples. Overall selectivity was established through determination of drug purity and resolution of the peak each time. Reproducibility of the method was established through separate studies on a mixture of degradation samples by different persons on the same chromatographic system as well as on different chromatographic systems on different days by another analyst. Various system suitability parameters were also evaluated using freshly prepared mobile phase each time.

 

RESULTS AND DISCUSSION:

Method development and optimization:

The peak of pure FEN obtained using 50 % methanol and 50 % 10 mM ammonium formate (v/v) suggested to employ 80 % methanol and 20 % 10 mM ammonium formate (v/v), as the mobile phase. Forced degradation samples were then analyzed using the same mobile phase flowing at a rate of 1.0 mL min1 on a C18 column employing DAD detection. The method resolved the drug and degradation products under acidic, basic, neutral, photolytic and oxidative conditions. To improve the peak shape and tailing, acetonitrile was introduced in the mobile phase and all other variables were kept the same. This mobile phase could optimally resolve FEN and all the degradation products formed under different conditions. The relative retention time (tR) of each peak of the degradation product with respect to fenoverine is given in Table I.

 

Validation of the method:

Linearity: Peak area and concentrations were subjected to the least square linear regression analysis to calculate the calibration equations and correlation coefficients. The calibration plot for FEN assay was linear over the calibration range 550 g mL1, and the regression coefficient, slope and intercept were 0.99770.00060, 26226.66993.04 and 133302.3316725 respectively. These results demonstrate an excellent correlation between the peak area and analyte concentration.

 

a)

 

b)

 

c)

 

d)

 

e)

 

f)

 

Fig. 2. HPLC chromatograms showing resolution of fenoverine and degradation products in: a) Standard chromatogram of fenoverine before degradation b) 1 mol L1 HCl at 70 C after 1 h, c) 0.01 mol L1 NaOH at RT after 1 h, d) water at 70 C after 5 h, e) sunlight after 5 h, f) 6 % H2O2 at RT after 24 h

 


TABLE I. RELATIVE RETENTION TIME, PEAK PURITY DATA AND SYSTEM SUITABILITY PARAMETERS OF FENOVERINE AND ITS DEGRADATION PRODUCTS

Parameter

Peak

DP-I

DP-II

DP-III

DP- IV

FEN

Relative retention time (tR)a

0.376

0.476

0.602

0.764

1.000

Peak purity index

0.999

1.000

0.999

0.999

1.000

Asymmetry (As)

1.1

1.4

1.2

1.2

1.3

Tailing factor (T)

1.1

1.3

1.1

1.1

1.3

Resolution (Rs)

2.5

4.0

4.7

5.12

5.02

Capacity factor (k)

1.5

2.17

3.0

4.08

5.65

Selectivity ()b

1.22

1.26

1.26

1.27

1.3

Theoretical plates (N)

5366

3593

6240

9465

6773

a With respect to FEN, b With respect to succeeding peak.

 


Limits of detection and quantification: LOD was 0.05 g mL1 for FEN at a signal--to-noise ratio of 3:1 and the LOQ was determined as 0.5 g mL1 for FEN at a signal-to-noise ratio of 10:1.

 

Precision: Intra-day precision was expressed through relative standard deviation of six repeated assays of samples at three concentration levels. Inter-day precision was determined by analyzing the same set of samples on three different days. RSD in the precision study for the FEN assay was less than 1.0 % and confirmed that the method was highly precise. Results of the precision study for FEN by the proposed RP-HPLC method are given in Table II.

 

TABLE II. PRECISION DATA OF THE PROPOSED RP-HPLC

Actual conc.

(mg mL1)

Measured concentration (mg mL1)a

Interday

Intraday

5.0

4.990.006

5.0020.02

20

20.050.07

20.050.04

40

40.0670.38

40.200.28

a Mean SD, n = 6.

 

Recovery: Standard addition method was used to examine the recovery of the RP-HPLC method. Recovery of FEN from bulk drug samples ranged from 100.080.3921 to 100.280.0777 and that from tablet dosage forms ranged from 100.571.692 to 101.400.6145

(Table III).

 

Table III. Recovery for bulk drug substance and drug product

Conc. of drug taken

(g mL1)

Conc. of standard added

(g mL1)

Conc. found

(g mL1)

Recovery (%)

 

Bulk drug substance

9.9516

5.0

14.960.0196

100.080.3921

9.9516

10.0

19.980.0078

100.280.0777

9.9516

15.0

24.970.0230

100.100.1533

Drug Product

9.936

5.0

14.990.0456

101.400.6145

9.936

10.0

19.990.1692

100.571.692

9.936

15.0

25.070.2173

100.871.448

Mean SD, n = 3

 

Specificity. There was no interference due to placebo, sample diluents and degradation products. Resolution between closely eluting degradation products, i.e., between DP-I, DP-II, DP-III, DP-IV and FEN were greater than 2.0, illustrated the chromatographic selectivity of the method.

 

Stability of stock solution. During solution stability and mobile phase stability experiments, RSD for the FEN assay was within 1% for three replicates. Results of the solution stability and mobile phase stability experiments confirmed that standard solutions and solutions in the mobile phase were stable for up to 48 h during the FEN assay.

 

Forced degradation:

During optimization of the hydrolytic degradation process, drug samples were initially placed in 1mol L1 HCl, 0.1 mol L1 NaOH and in water at 70C. However, after 12 and 24 h, the FEN sample in HCl and water showed 80 and 50 % degradation, respectively, while FEN in 0.1 mol L1 NaOH was completely degraded. Thus, we decided to carry out degradation at room temperature for the 0.1 mol L1 NaOH sample. The compound was completely degraded to DP-IV instantly at room temperature. When NaOH concentration was lowered to 0.01 mol L1, the sample containing FEN was 54% degraded at room temperature after 1h. Four degradation products (DP-I, DP-II, DP-III and DP-IV) were present in these forced samples, DP-IV being the major one (Fig. 2c). Degradation pattern under all forced conditions except oxidation and thermal showed the presence of DP-IV, which is the major degradation product of FEN formed during every degradation study. As illustrated in Fig. 2a, 2b, 2c and 2d for acidic, alkaline, neutral hydrolysis and photolytic degradation, DP-IV was observed in reasonable amounts. Degradation in neutral hydrolytic medium at 70C (Fig. 2c) occurred at a slower rate than under other hydrolytic conditions. Neutral hydrolysis showed two degradants, DP-III and DP-IV while oxidative degradation produced two degradants, DP-I and DP-II (Fig. 2e). No degradation was seen in solid drug kept at 90 C for 2 days. Results proved that the degradation of FEN was more pronounced under basic hydrolytic condition than under acidic and neutral conditions. Summary of the data for all forced degradations is given in Table IV. Peak purity test suggested that the FEN peak as well as the peaks of FEN degradation products were pure for all the forced samples analyzed (Table I). No additional peak was observed in chromatograms obtained for the extended runtime of 25 min in a sample study.

 

Chromatogram of pure FEN was compared with that of the drug subjected to the different forced conditions; all the chromatograms showed changes in the retention time. Peak purity of all the degradation products was found to be 0.9999 to 1.0, which confirm the purity of the products.


TABLE IV. SUMMARY OF FORCED DEGRADATION STUDY RESULTS

Stress Condition

Time

FEN (%)

Major degradation products

Acid hydrolysis (1 mol L1 HCl, 70 C)

1h

45.7

DP-IV

Basic hydrolysis (0.01 mol L1 NaOH, 70 C)

1h

46.32

DP-IV

Aqueous hydrolysis (70 C)

5h

34.3

DP-III and DP-IV

Oxidation (3% H2O2, RT)

24h

84.2

DP-I and DP-II

Thermal (90 C)

18h

97.4

No degradation product formed

Sunlight

5h

86.0

DP-IV

 


The goal of determining FEN in the presence of degradation products by the proposed stability indicating RP-HPLC method was successfully achieved but the method can be also used for routine quality control of capsules.

 

CONCLUSIONS:

The RP-HPLC method developed for quantitative analysis of fenoverine in both bulk drug and pharmaceutical dosage form is precise, accurate and specific. Satisfactory results were obtained by validation of the method. No attempt was made to characterize and quantify the degradation products; quantitation is possible after isolation of degradation products in pure form. This method can be used for the estimation of fenoverine in the presence of degradation products obtained under different forced conditions.

 

ACKNOWLEDGEMENTS:

Authors would like to thank Microlabs Pharmaceuticals Ltd., Bangalore, India for providing the gift sample of fenoverine.

 

REFERENCES:

1.        ICH(Q1AR2) , Harmonized Tripartite Guideline,Stability testing of New drug substances and products, In : Proceedings of the International Conference on Harmonization, Geneva. Aug. 2003.

2.        Mironneau J, Arnaudeau S and Mironneau C. Fenoverine inhibition of calcium channel currents in single smooth muscle cells from rat portal vein and myometrium. Br. J. Pharmacol. 1991; 104:65-70.

3.        Suresh B et al. High Performance Liquid Chromatographic Determination of Fenoverine in Human Serum: Application to Pharmacokinetic Study. J. Liq. Chrom. Rel. Technol. 2008; 31: 2101 2112.

4.        Robert R, Alain A and Patrick C. Determination of fenoverine in tissue samples by high-performance liquid chromatography. J Chromatogr B Biomed Sci. 1994; 654 : 159-163.

5.        Hu OY et al. Determination of fenoverine, a modulator of smooth muscle motility, in capsules and in human plasma: application to dosage form stability and a pilot study in humans. J Pharm Sci. 1992; 81:91-93.

6.        International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedure: Text and Methodology Q2 (R1), Current Step 4 version, ICH Geneva, Nov. 2005.

 

 

 

 

Received on 26.08.2010 Modified on 03.09.2010

Accepted on 11.09.2010 RJPT All right reserved

Research J. Pharm. and Tech. 4(2): February 2011; Page 237-241