INTRODUCTION:

Fenpiverinium bromide (FPB) is Antispasmodic and Antimuscarinic. Chemically it is 2,2-Diphenyl-4-piperidinobutyramidemethylbromide with its molecular formula of C₂₂H₂₉BrN₂O and its molecular weight is 417.4. Extraction Spectrophotometry method¹, UV-visible spectrophotometry² were described for Fenpiverinium bromide in literature. There was no Liquid chromatography analytical method was mentioned in latest issues of official pharmacopoeias^3-7 like European, Indian, USP etc.But only in Martindale Pharmacopoeia⁸, we find some details of Fenpiverinium bromide but it did not describe an analytical procedure so we proposed the present work to quantitatively estimate the low content of Fenpiverinium bromide in Spasgan® tablets. Each tablet contains Metamizole sodium (500mg), Pitofenone HCL (5mg) and Fenpiveriniumbromide (0.1mg).This Paper describes the reliable method to quantify only the low content of Fenpiverinium bromide in Spasgan tablets. The RP-HPLC method reported in this study was validated in accordance with the International Conference on Harmonization (ICH) guideline ^9-12.

MATERIAL AND METHOD:

Chemicals and Reagents:

Methanol(HPLCgrade),Sodiumdihydrogenphosphatemono-hydrate, Triethylamine, Orthophosphoricacid, HCl, Sodiumhydroxide and Hydrogen Peroxide are all AR grade were procured from Merck India. Milli- Q water was used. Fenpiverinium bromide working standard (percentage purity 99.5) and Spasgan tablets is obtained from Wockhardt, Aurangabad, India.

Instrumentation:

The Waters High performance liquid chromatographic system used was equipped with a solvent delivery Waters 2496 series HPLC Pump, a degassing device, a 20µl injection loop (Rheodyne injector). Waters with PDA/UV detector and controlled by Empower-2 software.

Chromatographic conditions:

The HPLC column of Supelcosil LC-CN (Sigma-Aldrich), 250mm×4.6mm, 5µm was used. The mobile phase of phosphate buffer and Methanol (66:34%v/v) of 40mM.Add 1.0ml of Triethylamine and mixed well. Sodiumdihydrogen phosphate having P^H(6.2±0.05) is adjusted with 1% ortho Phosphoric acid was used as a mobile phase.Then mobilephase was filtered through 0.45µ membrane filter and degas. At Ambient column oven temperature with 1ml/min flow rate and detection wavelength of 210nm having injection volume of 20µl was used.

Preparation of standard solution:

Weighed accurately about 20.0mg of Fenpiverinium bromide working standard and transferred in to 100ml volumetric flask. Then added 70ml of methanol, sonicated to dissolve and make up to volume with methanol and mixed well. Again transferred 5ml of the above solution in to 100ml volumetric flask and added 10ml of milli-Q water, then finally diluted up to the mark with methanol and mixed well. Then filtered the solution through 0.45µ membrane filter.

Calibration curve:

The calibration curves were prepared by taking 70% to 130% from stock solutions to obtain final concentrations of 7.01, 8.01, 9.01, 10.01, 11.01, 12.01, 13.01 µg/ ml of FPB with mobile phase. The calibration curve Fig No-1and 2 was plotted by average peak area against concentration and regression equation was computed.

Fig;1 Calibration curve of FPB in presence of Standard.

Fig;2 Calibration curve of FPB in presence of Placebo.

Fig (3) A typical HPLC-PDA/UV detector chromatogram of FPB Sample.

Fig (4) A typical HPLC-PDA/UV detector chromatogram of FPB Standard.

Preparation of sample solution:

Weigh 20 tablets and transfer (equivalent to about 2 mg of Fenpiveriniumbromide) in to a 200 ml volumetric flask then added 20 ml of milli-Q Water and sonicated for 5 mins. Then added 140 ml of methanol and sonicated for 15 mins with intermediate shaking. Cooled to room temperature and diluted to volume with methanol and mixed well. Filtered the solution through 0.45µ membrane filter.

High Performance liquid chromatographic analysis:

A Simple isocratic high Performance liquid chromatographic method was developed for the determination of FPB in presence of Metamizole sodium (MTM) and Pitofenone Hydrochloride (PTF). To optimize the HPLC assay parameters, mobile phase conditions (buffer: organic phase), type of column and its dimensions and choice of detection wavelength were investigated. The mobile phase was selected after several trials. The tailing of the peak was reduced dramatically by addition of triethyl amine. Good resolution was obtained with a mobile phase containing of Sodium dihydrogen phosphate Buffer (40mM ) and methanol in the ratio 66:34 v/v. various types of stationary phase with different dimensions and particle size were used. It was found that Supelcosil LC-CN column (250mm×4.6mm, 5µm) gave the most suitable resolution. With the optimized chromatographic conditions, a steady baseline was recorded.

RESULTS AND DISCUSSIONS:

To develop a precise, accurate and suitable HPLC method for the Quantitative estimation of FPB different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination.

Linearity and Range:

Linearity test solutions for assay method were prepared from stock solution at seven different concentration levels from 70 to 130% of assay analyte concentration (6.98, 7.98, 9.02, 10.04, 10.93, 11.92, 12.93 μg/ml). The peak area versus concentration data was performed by least-squares linear regression analysis. The linear fit of the system was illustrated graphically. The calibration curve for FPB standard and in presence of placebo were constructed using the peak-area versus the nominal concentrations of the analytes. The slope and regression coefficient of the calibration curve demonstrates that the method has adequate sensitivity. Also the method shows good linear response of Linearity and Range in presence of placebo in the same range of 70% to 130% of test concentration with their results are presented in Table-1.

Table-1: Linear regression data for FPB(n=6).

Linear regression Data	FPB in Standard	FPB in Placebo
Concentration range (μg/ml)	6.98-12.93	6.93-12.95
Regression equation	Y=58531X-32166	Y=57274X-6924.3
Standard error of Slope	58530	57273.71
Correlation Co-efficient	0.9998	0.9996
Steyx	2428.027	2661.49
LOD(μg/ml)	0.137	0.153
LOQ(μg/ml )	0.415	0.465

Accuracy (Recovery studies):

The accuracy was determined by Recovery experiments. The method was performed by in triplicate by standard addition method of adding Fenpiverinium bromide working standard to placebo in the range of 70% to 130% of test concentration. The results are summarized in Table-2.

Table-2: Recovery of FPB solution (n=3).

S.NO	%	Stdadded (mg)	Std Recovered (mg)	% Recovery
1	70	1.3857	1.3840	99.88
2	80	1.5884	1.5869	99.87
3	90	1.7725	1.7870	100.25
4	100	1.9797	1.9912	100.58
5	110	2.1938	2.1989	99.78
6	120	2.3852	2.3893	100.17
7	130	2.5893	2.6168	101.06
MEAN				100.23
SD				0.460
%RSD				0.5

SD-Standard deviation, RSD-Relative standard deviation

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

For determining the limits of detection (LOD) and Quantitation (LOQ), the method based on the residual standard deviation (SD) of a regression line and slope was adopted. To determine the LOD and LOQ, a specific calibration curve was studied using samples containing the analytes in the range of the detection and quantitation limits. The LOD and LOQ results were shown in table-1

Repeatability and Ruggedness:

The repeatability of the assay method is established by estimating the assay for 6 different sample preparations of the same batch. The ruggedness of the assay method (Table-3) was assessed by estimating the assay of 6 different sample preparation of the same batch by different analyst on a different HPLC system using column of different lot number and on a different day. The results are statistically valid .

Table-3: Repeatability and Ruggedness % RSD (n=6) for FPB.

S. no	Instrument	Analyst	%Assay	Mean	SD	% RSD
1	Instument-1	Analyst-A	101.10	101.00	0.415	0.4
			101.20
			100.40
			101.50
			100.60
			101.20
2	Instument-2	Analyst-B	100.20	100.75	0.834	0.8
			101.90
			100.80
			100.00
			101.60
			100.00

Selectivity:

The selectivity of the assay method is established by injecting blank (Diluent), placebo, standard and sample of Fenpiverinium bromide preparation, in to the chromatograph. It was observed that there is no interference from the peaks obtained for the chromatograms of blank and placebo with that of Fenpiverinium bromide peaks obtained for the chromatogram of sample and standard (fig no-3 and 4) of Fenpiverinium bromide preparation. Hence the proposed method is higly selective and specific.

Degradation Studies:

The forced degradation studies were performed to establish the stability indicating nature of the assay method. The peaks obtained by stress conditions like Acid (5N HCl), Alkali (5N NaOH), Peroxide (3%v/v H₂0₂), UV light (254nm), Thermal (105C).The peak purity results were summarized in Table-4.The degradation peaks are well separated from Fenpiverinium bromide peaks and the purity angle was less than purity threshold in all chromatograms was inferred from Chromatograms of fig no-5 to 9.

Acidic degradation studies:

Add 5ml of 5N HCl was added to 10ml of drug solution to get final concentration of 10 mcg/ml of FPB. This solution was refluxed at 70^˚c for 10minutes.

Basic degradation studies:

Add 5ml of 5N NaOH was added to 10ml of drug solution to get final concentration of 10 mcg/ml of FPB. This solution was refluxed for 5minutes.

Oxidative studies:

Add 5ml of 3% v/v H₂O₂was added to 10ml of drug solution to get final concentration of 10 mcg/ml of FPB. This solution was refluxed for 5minutes.

UV Studies:

The drug solution of final concentration of 10 mcg/ml of FPB was exposed to UV light at 254nm for 5 days.

Temperature stress studies:

The drug solution of final concentration of 10 mcg/ml of FPB was maintained at 105˚c for 8 hours.

Table-5Robustness for FPB

S. no	Parameter	Condition	% Assay	Tailing	Theoretical plates	%RSD
1	Flow rate (ml/min)	0.9	100.40	1.1	7769	0.9
		1.0	100.00	1.1	7126	0.8
		1.1	100.10	1.1	7034	1.7
2	Wavelength (nm)	205	99.90	1.1	6958	0.4
		210	99.60	1.1	6907	0.6
		215	98.80	1.1	6985	1.0
3	pH of Buffer	6.0	101.20	1.1	6921	0.5
		6.2	99.90	1.1	6907	0.6
		6.4	99.60	1.1	6997	0.6
4	% Organic in mobile phase	61:29	98.60	1.2	6876	0.4
		66:34	100.50	1.2	7099	0.8
		71:39	98.50	1.2	6853	0.8

Robustness:

The robustness of the assay method is assessed by deliberately modifying the chromatographic conditions like fiow rate (±1 mL), wavelength (±5 nm), P^H of buffer in mobile phase (±0.2)., mobile phase composition(±5%v/v). The assay results (Table-5) obtained in the range between 98.50 to 101.20% which were close to precision results. Hence the method is highly robust.

System Suitability:

The system suitability (Table-6) was determined based on their column efficiency value of more than 2000 theoretical plates and the tailing factor of less than 2.0 was obtained. For system suitability studies, five replicate injections of FPB were injected and the parameters like RSD of peak area ratio, column efficiency and tailing factor of the peaks were calculated. Results are shown in Table 6.

Table -6 system suitability for FPB (n=5).

S. no	Parameters	Tailing factor	Theoretical plats	%RSD
1	Repeatability	1.1	7230	0.3
2	Ruggedness	1.2	7769	0.9
3	Linearity	1.1	7512	0.4
4	Accuracy	1.2	6994	0.2
5	Stability	1.3	5850	0.5
6	Filter recovery	1.3	5694	0.2
7	Specificity	1.2	6680	0.9

Solution Stability:

The stability of working standard and sample solution was established at periodic intervals up to 24 hours by keeping the Auto sampler temperature at room temperature (25ºc).It shows that working standard and sample solution were stable up to 22 hours at room temperature (25ºc).

CONCLUSION:

The developed chromatographic assay fulfilled all the requirements to be identified as reliable method, including accuracy, linearity, recovery and precision data. It is a highly specific and precise analytical procedure. Therefore, this HPLC method can be used as a routine sample analysis as well as stability testing. The developed method is stability-indicating and can be used for quantifying FPB of very low content in combination with high content of Metamizole sodium and Pitofenone HCl in tablet dosage form.

REFERENCES:

1. Vladmir Kubicek, et al. Determination of low contents of Fenpiverine Bromide by extraction spectrophotometry. Microchim Acta. 2003; 142:273-276.

2. Shubhangi Daswadkarl, Jagdish Gaikwadi and Pinal Mauryal. Analytical method development of Fenpiverinium bromide by UV-Visible spectrophotometry. Int. J. Drug Formulation and Research.2011; 2: 249-254.

3. The United States Pharmacopoeia 24, Twin brook Parkway, Rockvile 2000, MD 20852.

4. Budavari S. The Merck index, Merck and Co. Press: Whitehouse Station, NJ, 12th Edn, 1997.

5. European Pharmacopoeia , 3rd edn., CEDEX Strasbourg,1997.

6. British Pharmacopoeia , HMSO Publication Centre, London.

7. Czech Pharmacopoeia , Grada Publishing, Praha,1997.

8. Reynolds J.E.F. Martindale, The Extra Pharmacopoeia, 31st ed, Royal Pharmaceutical society: London, 1996; 1706.

9. Validation of analytical procedures; Text and Methodology Q2 (R1), International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human,Geneva,Switzerland,2005.

10. Shabir GA, Validation of HPLC methods for pharmaceutical analysis: Understanding the differences and similarities between validation requirements of the U.S. Food and Drug Administration, the U.S Pharmacopoeia and the International Conference on Harmonization. J. chromatogr A 2003;987:57-56.

11. Shabir GA, Lough WJ, Shafique AA, Bradshaw TK, Evaluation and application of best practice in analytical method validation. J. Liq Chromatogr Relat Technol 2007;30:311-333.

12. ICH, Q2A, Textbook on Validation of Analytical Procedures, International Conference on Harmonization, Geneva, October, 1994.

Received on 22.08.2011 Modified on 08.09.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1777-1781

Stress condition	Drug Substances			Drug products
Stress condition	%Degradation	Purity angle	Purity threshold	%Degradation	Purity angle	Purity threshold
Acid Degradation (5N HCL)	5.23	0.3356	0.6099	3.52	0.2295	0.5012
Alkali Degradation (5N NaOH)	15.64	0.3212	0.5473	17.63	0.5603	0.5908
Oxidative Degradation (3%v/v H₂0₂)	9.76	0.2985	0.5309	11.16	0.3135	0.5554
UV light Degradation 254nm for 5Days	1.68	0.2535	0.4846	1.88	0.2965	0.4559
Heat Degradation (105⁰C for 8Hours	2.83	0.2430	0.4970	6.27	0.3222	0.5241