Estimate Hyoscine Butylbromide and Mefenamic Acid by RP-HPLC Method Development and Accelerated Stability Study in Pure and Combine Dosage Form

The aim of the present study was to develop and validate stability indicating HPLC method for simultaneous estimation of Hyoscoine butyl bromide (HBB) and Mefenamic acid (MEF). HPLC method for simultaneous analysis of both drugs was developed and validated according to ICH guideline. Efficient chromatographic separation was achieved on ODS column C18 (250 mm × 4.6 mm, 5 μm) using the optimized mobile phase. Stability indicating assay method was carried out by different stress degradation conditions. In HPLC method, the Retention time for HBB and MEF was 3.21 and 5.07 min using optimized mobile phase potassium dihydrogen phosphate buffer (pH 5.0) and methanol (60:40 % v/v) with a flow rate of 1 ml/min. The multiple wavelength UV detector was set at a 237 nm for measurement of all compound. Quantification based on measuring the peak areas. The degradation of HBB, MEF and Formulation was shown to be highest in alkaline condition. Linearity was observed in concentration range of 13μg/ml and 12.5-37.5 μg/ml for HBB and MEF respectively. All validation parameters were within the acceptable range. Moreover, the % RSD for repeatability, inter and intraday precision was found to be within the range, which reveals that the method is precise. Accuracy study of the drug in marketed preparation also report in the limit. Assay of the dosage form finalized the applicability of this method for estimation of Hyoscine Butylbromide and Mefenamic Acid tablet dosage form.

The combination formulation is used as an anti spasmodic analgesic for the spasm in GIT and UTI.Literature study reveals that there are number of method published for the HBB alone and in combination with other drugs like UV [5][6][7], HPLC [8,9], stability indicating HPLC [10], LC-MS [11].For MEF UV [12,13], HPLC [14][15][16][17], HPTLC [18], stability indicating HPLC [19,20] are reported in single and in combination with other drugs.There are no any single method was found for the estimation of these two drugs in combination.Both drugs are official in IP-2014 [21], BP-2009 [22], USP30-NF25 [23].The present work, a successful attempt has been made to estimate both these drugs simultaneously using stability indicating RP-HPLC method in different conditions.This study attempts to

Tandel et al. / Stability RP-HPLC HBB and MEF
2 / 16 develop a simple, accurate and precise analytical chromatographic method, which can quantify these drugs simultaneously from a combined tablet dosage form.The developed method was validated as per ICH guidelines and found to comply with the acceptance Criteria [24,25].

Materials and Reagents
MEF was procured from Triveni Interchem Pvt Ltd Vapi and HBB was procured from Sovereign Pharma Pvt Ltd Daman as gift sample.All the reagents of HPLC grade including Water, Methanol, Acetonitrile were procured from Finar.NaOH, H2O2, dihdrogen phosphate and HCl were procured from Specrtochem.The Pharmaceutical formulation used in this study was Hyoscimax-MF Tablet procured from the local market and labeled to contain 20mg HBB and 250 mg MEF per Tablet.

Preparation of mobile phase
Composition: Potassium dihydrogen phosphate buffer (pH 5.0): Methanol (60:40 v/v) Preparation of Buffer: Take 6.8 gm of Potassium dihydrogen phosphate buffer and add into the 800 ml of water and shake well to dissolve the KH2PO4 and then add 200 ml of water to make 1000 ml of Buffer Solution.Adjust the pH of Buffer with the 0.1 N NaOH to make the pH of Buffer 5.0

MEF standard stock solution: (250 μg/ml)
A 250 mg of MEF was weighed and transferred to a 100 ml volumetric flask and volume made up by methanol (2500 μg/ml).Take 10 ml from this solution in 100 ml volumetric flask and volume was made up to the mark with methanol.

HBB standard stock solution: (20 μg/ml)
A 200 mg of HBB was weighed and transferred to a 100 ml volumetric flask and volume was made up to the mark with methanol (2000 μg/ml).Take 10 ml from this solution in 100 ml volumetric flask and make up with  methanol (200 μg/ml).From the above solution take 10 ml in 100 ml volumetric flask and make up the volume with methanol.

Preparation of standard solution of binary mixtures of MEF (25 μg/ml) and HBB (2 μg/ml)
Take 1 ml from the MEF stock solution and 1ml from HBB stock solution and transferred to 10 ml volumetric flask and volume made up to the mark by methanol.

Preparation of Sample Solution
Take Tablet Powder equivalent to 250 mg of MEF and 20 mg of HBB was transferred to a 100 ml volumetric flask and make up 25 ml with Mobile phase shake well and Sonicate for 15 minutes and finally make up the volume up to 100 ml.The solution was filtered through Whatman filter paper no.42.Take 1 ml from this and transferred to 10 ml volumetric flask and made up volume up to the mark with mobile phase.[MEF25 μg/ml and HBB2 μg/ml] The solution was injected 20 µl.The areas of resulting peak were measured at 237nm.

Selection of Elution Mode
Reverse phase chromatography was chosen because of its recommended use for ionic and moderate to nonpolar compounds.Reverse phase chromatography is not only simple, convenient but also better performing in terms of efficiency, stability and reproducibility.C18 column is least polar compare to C4 and C8 columns.Here, A 250 x 4.6 mm column of 5.0μm particle packing was selected for separation of MEF and HBB.Isocratic mode was chosen due to simplicity in application and robustness with respect to longer column stability.

Selection of Wavelength
Standard solution of MEF (25 μg/ml) and Standard solution of HBB (2 μg/ml) were scanned between 200-400 nm using UV-visible spectrophotometer.Both solutions were scanned between 200-400 nm.Wavelength was selected from the overlay spectra of above solutions.

System Suitability Test
It is an integral part of chromatographic method.These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed.System suitability tests are based on the concept that the equipment, electronics, analytical operations and samples constitute an integral system that can be evaluated as a whole.System suitability testing provides assurance that the method will provide accurate and precise data for its intended use.
Fornulas for calculation of SST are, where,   is resolution  1 and  2 are the retention times of components 1 and 2,  1 and  2 are peak width of components 1 and 2.

Theoretical plate
where, is no of theoretical plate.
is the retention time.

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Tailing factor  = 0.052 where,  is tailing factor 0.05 is with of peak at 5% height and f is distance at 5% height.

Acid degradation
Acid decomposition studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask. 2 ml of 0.1 N HCl solutions was added and mixed well and put for 4 hrs.After time period the volume was adjusted with diluent to get 2 μg/ml for HBB and 25 μg/ml for MEF.

Base degradation
Basic decomposition studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask. 2 ml of 0.1 N NaOH solutions was added and mixed well and put for 4 hrs.After time period the volume was adjusted with diluents to get 2 μg/ml for HBB and 25 μg/ml for MEF.

Oxidative degradation
Oxidative decomposition studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask. 2 ml of 3% H2O2 solutions was added and mixed well and put for 4 hrs.After time period the volume was adjusted with diluent to get 2 μg/ml for HBB and 25 μg/ml for MEF.

Photo degradation
Photo Degradation studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask.The volumetric flask was keep in presence of Sunlight for 48 h.Then the volume was adjusted with diluent to get 2 μg/ml for HBB and 25 μg/ml for MEF.

Thermal degradation
Thermal Degradation studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask.The volumetric flask was stored in oven at 110°C for 4 h.Then the volume was adjusted with diluent to get 2 μg/ml for HBB and 25 μg/ml for MEF.

Linearity
The linearity for MEF and HBB were assessed by analysis of combined standard solution in range of 12.5-37.5μg/mland 1-3 μg/ml respectively.5, 7.5, 10, 12.5,15 ml solutions were pipette out from the Stock solution of MEF (250 μg/ml) and HBB (2 μg/ml) and transfer to 100 ml volumetric flask and make up with mobile phase to obtain 12.5,18.75,25,31.25 and 37.5 μg/ml and 1,1.5,2,2.5 and 3 μg/ml for MEF and HBB respectively In term of slope, intercept and correlation co-efficient value, the graph of peak area obtained verses respective concentration was plotted.

Precision
Results should be expressed as Relative standard deviation (RSD) or coefficient of variance.

A. Repeatability
Middle concentration from calibration curve of MEF and HBB was injected six times and areas of peaks were measured and %R.S.D. was calculated.

B. Intra-day precision
Standard solution containing lower, middle, higher concentration range of MEF and HBB were analyzed three times on the same day and %R.S.D was calculated.

C. Inter-day precision
Standard solution containing lower, middle, higher concentration range of MEF and HBB were analyzed three times on the different day and %R.S.D was calculated.

Accuracy
 For MEF 17 µg/ml drug solution was taken in three different flasks label A, B and C. Spiked 80%, 100%, 120% of standard solution in it and diluted up to 100ml.The area of each solution peak was measured at 237 nm.The amount of MEF was calculated at each level and % recoveries were computed.
 For HBB 1.3 µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80%, 100%, 120% of standard solution in it and diluted up to 100ml.The area of each solution peak was measured at 237 nm.The amount of HBB was calculated at each level and % recoveries were computed.

LOD and LOQ
The LOD was estimated from the set of 6 calibration curves used to determination method linearity.The LOD may be calculated as,

Robustness
Following parameters were changed one by one and their effect was observed on system suitability for standard preparation.

Analysis of marketed formulation by developed method
Take Tablet Powder equivalent to 250 mg of MEF and 20mg of HBB was transferred toa 50ml volumetric flask and make up 25 ml with Mobile phase shake well and Sonicate for 15 minutes and finally make up the volume up to 50 ml.The solution was filtered through Whatman filter paper no.42.Take 1 ml from this and transferred to 10 ml volumetric flask and made up volume up to the mark with mobile phase.[MEF 25 μg/ml and HBB 2 μg/ml] The solution was injected 20 µl.The areas of resulting peak were measured at 237 nm.

Selection of Wavelength
Both MEF and HBB show reasonably good response at 237nm.Shown in Figure 3.

Selection of Mobile Phase
After considering the varying combinations of various mobile phases, Phosphate Buffer: Methanol (60:40 % v/v pH 5).Take 6.8 gm KH2PO4 into a 1000 ml beaker, add 800 ml water and dissolve it adjust pH 5 with OPA.Make up Volume 1000 ml with water was finalized as it was showing good peak shapes and a significant amount of resolution.Final chromatogram of HBB and MEF are shown in Figure 4.
System suitability Parameters-Result of system suitability test are shown in Table 1.

Acid degradation
Acid decomposition studies were performed with 2 μg/ml of HBB and 25 μg/ml of MEF.Chromatogram for the studies were given in Figures 5-8.

Base degradation
Basic decomposition studies were performed with 2 μg/ml of HBB and 25 μg/ml of MEF.Chromatogram for degradation were shown in Figures 9-12

Oxidative degradation
Oxidative decomposition studies were with 2 μg/ml for HBB and 25 μg/ml for MEF.Chromatogram for degradation were shown in Figures 13-16.

Result for the % Degradation
Data for the %Degradation of HBB and MEF in standard as well as in sample was shown in Table 2.  28) Calibration graphs were constructed by plotting the peak area versus their corresponding concentrations (Figure 29 and 30).Good linearity was obtained in the range of 12.5-37.5μg/mland 1-3 μg/ml for MEF and HBB respectively.The results are shown in Table 3 and 4. LOD and LOQ were calculated from the slope and standard deviation of y-intercepts of the regression line of the calibration curve.The results are shown in Table 5.The precision of the method and instrument precision was evaluated and relative standard deviation (RSD) values were calculated.The RSD values for MEF and HBB showed that the precision of the method was satisfactory.The results are shown in Table 5.The accuracy of the method was determined by recovery studies.The recoveries were close to 100% for MEF and HBB.The results are shown in Table 6.Developed method was found to be robust when the mobile phase ratio, flow rate and pH was changed.The results are shown in Table 7 and 8

CONCLUSION
Forced degradation study of HBB and MEF was performed by RP-HPLC method in which Maximum degradation of MEF in base were found 18.33% for standard and 18.97 % for sample.And maximum degradation of HBB in thermal were found 20.06 % for standard and 20.78 for sample.The proposed sensitive RP-HPLC method gives accurate and precise results for determination of MEF and HBB in marketed formulation (tablet) without prior separation and is easily applied for routine analysis.The most striking feature of the method is its simplicity and rapidity.Method validation has been demonstrated by variety of tests for linearity, accuracy, precision, LOD, LOQ and robustness.The proposed method was successfully applied to determination of these drugs in commercial tablets.

Figure 2 .
Figure 2. Chemical Structure of Mefenamic acid

LOD = 3 . 3 ×
(SD/Slope) where, SD= Standard deviation of Y-intercepts of 6 calibration curves.Slope = Mean slope of the 6 calibration curves.The LOQ was estimated from the set of 6 calibration curves used to determine method linearity.The LOQ may be calculated as, LOQ = 10 × (SD/Slope) where, SD = Standard deviation of Y-intercepts of 6 calibration curves.Slope = Mean slope of the 6 calibration curves.

Figure 3 .
Figure 3. Overlay UV Spectrum of MEF and HBB showing selection of wavelength detection

Figure 8 .
Figure 8. HBB and MEF Acid Degradation Sample

Table 1 .
Results for system suitability test

Table 2 .
% Degradation of HBB and MEF

Table 3 .
Linearity data for MEF

Table 5 .
Precision and LOD, LOQ parameters for MEF and HBB

Table 6 .
Accuracy data of MEF and HBB

Table 7 .
Robustness data for MEF

Table 8 .
Robustness data for HBB

Table 9 .
Analysis of Marketed Formulation